Hemoproteins
occur widely in higher organisms and carry out very diverse functions. They all
contain the heme prosthetic group and can be categorized in terms of their
reactions with oxygen. These proteins are responsible for hydrogen peroxide
utilization and decomposition, oxygen transport and storage, electron
transport, oxygen reduction, and hydrocarbon oxidation. In order to explore the
diverse functions of hemoproteins, microperoxidases (MPs) are used to mimic the
active site of hemoproteins. MPs are water-soluble heme species that have been
used as models for the hemoproteins, and these MPs with various peptides (MP-5,
MP-6, MP-8, MP-9, & MP-11) were obtained by digestion of cytochrome c with
different proteases. In
this study, we showed that MP catalyzed
catalase-type reaction in addition to peroxidase-type and cytochrome P450-type
reactions. The relative catalytic specificities of Ac-MP-8 with a histidyl
ligand exhibit the following orders: peroixdase-type > cytochrome P450-type
> catalase-type reactions. The fifth ligands of native peroxidase,
cytochrome P450 and catalase are histidine, cysteine and tyrosine,
respectively. Therefore, we expressed the cytochrome c in E. Coli to mutate the fifth ligands of MPs to minic the effects of
proteins on the functions of hemoproteins. The expression of cytochrome c was
achieved by constructing the genes of iso-1-cytochrome c and heme lyase from Saccaromyces
cerevisiae in parallel into vector
pET-21b. In addition, the H23Y, H23C, H23R, L20K, Q21K and L20RQ21R mutants of
cytochrome c have been obtained to mimic the active site of hemoproteins. The
His-Ac-MP-11, Cys-Ac-MP-11, and Tyr-Ac-MP-11 will be
prepared from these mutants and served as the models for peroxidase, catalase,
and cytochrome P450, respectively. Effects of fifth ligands on the peroxidase,
catalase, and monooxygenase activities of these heme models will provide us the
structure-function relationships of heme enzymes and elucidate the diverse
functions of hemoproteins.